UGUS, a reporter for use with destabilizing N-termini.
Due to constraints in vector construction, reporter polypeptides often carry N-terminal sequences of extraneous origin. Since protein half-life can be influenced by small determinants in the N-terminus, such foreign sequences can destabilize proteins and compromise results of reporter-based studies. We provide a real-life example of this problem (destabilizing sequences derived from a ribosomal protein) and show that it can be solved with the ubiquitin fusion technique, in which ubiquitin sequences are placed upstream of the reporter, in our case beta-glucuronidase. Post-translational processing by characterized pathways removes the ubiquitin together with destabilizing sequences, generating a stable reporter whose N-terminus is constant.
Zaccaria Roberta Greco
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D
Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Ospedale S.Luigi, 10043 Orbassano, Torino, Italy.
MacWilliams
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H
Bozzaro
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S
Ceccarelli
A
A
D000818:Animals; D019677:Artificial Gene Fusion; D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D004023:Dictyostelium / Q000235:genetics; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014452:Ubiquitins / Q000235:genetics*
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D005966:Glucuronidase